Transcription Factor Runx2 Research Articles

Guideline for design of substrate stiffness for mesenchymal stem cell culture based on heterogeneity of YAP and RUNX2 responses.

Mesenchymal stem cells (MSCs) have the potential for self-renewal and multipotency to differentiate into various lineages. Thus, they are of great interest in regenerative medicine as a cell source for tissue engineering. Substrate stiffness is one of the most extensively studied exogenous physical factors; however, consistent results have not always been reported for controlling MSCs. Conventionally used stiff culture substrates, such as tissue-culture polystyrene and glass, enhance nuclear localization of a mechanotransducer YAP and a pre-osteogenic transcription factor RUNX2, and bias MSCs towards the osteogenic lineage, even without osteogenic-inducing soluble factors. The mechanosensitive nature and intrinsic heterogeneity present challenges for obtaining reproducible results. This review summarizes the heterogeneity in human MSC response, specifically, nuclear/cytoplasmic localization changes in the mechanotransducer yes-associated protein (YAP) and the osteogenic transcription factor RUNX2, in response to substrate stiffness. In addition, a perspective on the intracellular factors attributed to response heterogeneity is discussed. The optimal range of stiffness parameters, Young's modulus, for MSC expansion culture to suppress osteogenic differentiation bias through the suppression of YAP and RUNX2 nuclear localization, and cell cycle progression is likely to be surprisingly narrow for a cell population from an identical donor and vary among cell populations from different donors. We believe that characterization of the heterogeneity of MSCs and understanding their biological meaning is an exciting research direction to establish guidelines for the design of culture substrates for the sophisticated control of MSC properties.

A new pathway for the transformation of AML in MDS: APA mechanism regulated by NUDT21 and RUNX1.

We have identified that NUDT21 plays a vital role in MDS transformations, while the transcription factor RUNX1 is essential for normal hematopoiesis, which is a high expression in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and we aim to explore the linkage between the two genes and new pathways for MDS transformation to AML. Prediction of RUNX1 expression levels and its relationship with NUDT21 in AML and MDS patients was performed using bioinformatics techniques and validated in patients. Using lentiviral packaging technology, NUDT21 knockdown and overexpression models were developed in AML and MDS cell lines. These models were validated using quantitative polymerase chain reaction (qPCR) and western blotting. The cell cycle, apoptosis, differentiation, and cytokines were examined by flow cytometry, CCK-8 analyzed proliferation, and the intracellular localization of NUDT21 and RUNX1 was examined by immunofluorescence. mRNA transcriptome sequencing was performed on THP-1, MUTZ-1, and Dapars analyzed SKM-1 cell lines and the sequencing data to observe the knockdown effect of NUDT21 on RUNX1. qPCR and western blot revealed a positive correlation between NUDT21 and RUNX1; both were located in the nucleus. Overexpression of NUDT21 reduced apoptosis, promoted cell proliferation, and possibly increased the invasive ability of cells. It also altered the APA site in the RUNX1 3'-UTRs region. NUDT21 regulates RUNX1 gene expression and promotes AML transformation in MDS through an APA mechanism.

Melatonin suppresses bone marrow adiposity in ovariectomized rats by rescuing the imbalance between osteogenesis and adipogenesis through SIRT1 activation

Accelerated imbalance between bone formation and bone resorption is associated with bone loss in postmenopausal osteoporosis. Studies have shown that this loss is accompanied by an increase in bone marrow adiposity. Melatonin was shown to improve impaired bone formation capacity of bone marrow-derived mesenchymal stem cells from ovariectomized rats (OVX-BMMSCs). To investigate whether the anti-osteoporosis effect of melatonin involves regulation of the equilibrium between osteogenic and adipogenic differentiation of osteoporotic BMMSCs. To induce osteoporosis, female Sprague-Dawley rats received ovariectomy (OVX). Primary BMMSCs were isolated from tibiae and femurs of OVX and sham-op rats and were induced towards osteogenic or adipogenic differentiation. Matrix mineralization was determined by Alizarin Red S (ARS) and lipid formation was evaluated by Oil Red O. OVX rats were injected with melatonin through the tail vein. Bone microarchitecture was determined using micro computed tomography and marrow adiposity were examined by histology staining. OVX-BMMSCs exhibited a compromised osteogenic potential and an enhanced lineage differentiation towards adipocytes. In vitro melatonin improved osteogenic differentiation of OVX-BMMSCs and promoted matrix mineralization by enhancing the expression of transcription factor RUNX2 in a dose-dependent manner. Moreover, melatonin significantly inhibited lipid formation and suppressed OVX-BMMSCs adipogenesis by down-regulating peroxisome proliferator-activated receptor γ (PPARγ). Intravenous injection of melatonin prevented bone mass reduction and bone architecture destruction in ovariectomized rats. Importantly, there was a significant inhibition of adipose tissue formation in the bone marrow. Mechanistic investigations revealed that SIRT1 was involved in melatonin-mediated determination of stem cell fate. Inhibition of SIRT1 abolished the protective effects of melatonin on bone formation by inducing BMMSCs towards adipocyte differentiation. Melatonin reversed the differentiation switch of OVX-BMMSCs from osteogenesis to adipogenesis by activating the SIRT1 signaling pathway. Restoration of stem cell lineage commitment by melatonin prevented marrow adipose tissue over-accumulation and protected from bone loss in postmenopausal osteoporosis. Determination of stem cell fate towards osteoblasts or adipocytes plays a pivotal role in regulating bone metabolism. This study demonstrates the protective effect of melatonin on bone mass in estrogen-deficient rats by suppressing adipose tissue accumulation in the bone marrow. Melatonin may serve as a promising candidate for the treatment of osteoporosis in clinics.

Integrin subunit β4 drives the aggressiveness of papillary thyroid carcinoma by enhancing MMP14 expression through transcription factor RUNX1 pathway

Integrin subunit β4 drives the aggressiveness of papillary thyroid carcinoma by enhancing MMP14 expression through transcription factor RUNX1 pathway

Intergenic CircRNA Circ_0007379 Inhibits Colorectal Cancer Progression by Modulating miR-320a Biogenesis in a KSRP-Dependent Manner.

Circular RNAs (circRNAs) are covalently closed RNA structures that play multiple roles in tumorigenesis and progression. Compared with exon‒intron circRNAs, the biological functions and implications of intergenic circRNAs in human cancer are still poorly understood. Here, we performed circRNA microarray analysis and identified an intergenic circRNA, circ_0007379, that was significantly downregulated in patients with colorectal cancer (CRC). The biogenesis of circ_0007379 was mediated by reverse complementary matches (RCMs) and was negatively regulated by the RNA helicase DHX9. Functionally, circ_0007379 suppressed CRC cell growth and metastasis in cell culture as well as in patient-derived organoid and xenograft models. Mechanistically, circ_0007379 acted as a scaffold to facilitate the processing of both pri-miR-320a and pre-miR-320a in a KSRP-dependent manner, leading to miR-320a maturation and subsequent repression of transcription factor RUNX1 expression. Thus, our findings establish a previously unrecognized function of circRNA in inhibiting CRC progression.

Plakophilin 3 facilitates G1/S phase transition and enhances proliferation by capturing RB protein in the cytoplasm and promoting EGFR signaling

Plakophilin 3 (PKP3) is a component of desmosomes and is frequently overexpressed in cancer. Using keratinocytes either lacking or overexpressing PKP3, we identify a signaling axis from ERK to the retinoblastoma (RB) protein and the E2F1 transcription factor that is controlled by PKP3. RB and E2F1 are key components controlling G1/S transition in the cell cycle. We show that PKP3 stimulates the activity of ERK and its target RSK1. This inhibits expression of the transcription factor RUNX3, a positive regulator of the CDK inhibitor CDKN1A/p21, which is also downregulated by PKP3. Elevated CDKN1A prevents RB phosphorylation and E2F1 target gene expression, leading to delayed S phase entry and reduced proliferation in PKP3-depleted cells. Elevated PKP3 expression not only increases ERK activity but also captures phosphorylated RB (phospho-RB) in the cytoplasm to promote E2F1 activity and cell-cycle progression. These data identify a mechanism by which PKP3 promotes proliferation and acts as an oncogene.

Regulatory Role of the RUNX2 Transcription Factor in Lung Cancer Apoptosis.

Lung cancer is the leading cause of cancer death globally. Numerous factors intervene in the onset and progression of lung tumors, among which the participation of lineage-specific transcription factors stands out. Several transcription factors important in embryonic development are abnormally expressed in adult tissues and thus participate in the activation of signaling pathways related to the acquisition of the tumor phenotype. RUNX2 is the transcription factor responsible for osteogenic differentiation in mammals. Current studies have confirmed that RUNX2 is closely related to the proliferation, invasion, and bone metastasis of multiple cancer types, such as osteosarcoma, breast cancer (BC), prostate cancer, gastric cancer, colorectal cancer, and lung cancer. Thus, the present study is aimed at evaluating the role of the RUNX2 transcription factor in inhibiting the apoptosis process. Loss-of-function assays using sh-RNA from lentiviral particles and coupled with Annexin/propidium iodide (PI) assays (flow cytometry), immunofluorescence, and quantitative PCR analysis of genes related to cell apoptosis (BAD, BAX, BCL2, BCL-XL, and MCL1) were performed. Silencing assays and Annexin/PI assays demonstrated that when RUNX2 was absent, the percentage of dead cells increased, and the expression levels of the BCL2, BCL-XL, and MCL1 genes were downregulated. Furthermore, to confirm whether the regulatory role of RUNX2 in the expression of these genes is related to its binding to the promoter region, we performed chromatin immunoprecipitation (ChIP) assays. Here, we report that overexpression of the RUNX2 gene in lung cancer may be related to the inhibition of the intrinsic apoptosis pathway, specifically, through direct transcriptional regulation of the antiapoptotic gene BCL2 and indirect regulation of BCL-XL and MCL1.

Transcriptomic Changes Associated with Rare Germline Missense ATM Variants in Chronic Lymphocytic Leukaemia

Background Germline pathogenic variants in ATM gene are associated with risk of chronic lymphocytic leukaemia (CLL) and poorer prognosis. However, genetic testing is revealing a large number of rare germline missense variants of uncertain significance (VUS) in ATM whose clinical value is unclear. In this study, we evaluated the impact of ATM germline missense variants on the CLL transcriptome. Methods The genetic germline and transcriptome data from 237 CLL cases were obtained from public CLL datasets (EGAD00001001464, EGAD0000100146612). Only rare (minor allele frequency < 0.5%) missense ATM germline variants annotated as VUS by Clinvar and Varsome databases and with pathogenic prediction by SIFT/PolyPhen were studied. The transcriptome analysis was carried out using the DEseq2, 3D RNA-seq and Enrichr tools. Adjusted p value (padj) < 0.15 was considered significant. Results Of the analysed dataset, 9 (3.8%) CLL cases carried rare germline missense predicted pathogenic VUS in ATM. Further analysis was performed within subgroups of patients based on their IGHV mutational status, as preliminary cluster analysis showed distinct gene expression profiles for these subgroups. In the IGHV mutated (m-IGHV) subgroup, six cases carried germline missense ATM VUS(s) (G204R, R717W, D817N, V1671G, T1871I, L2293R, L2293V), four had solo del(11q) and 77 were without any somatic and/or germline disruption in genes associated with DNA damage response (DDR), nor did they carry mutations in a panel of 10 recurrently mutated genes (TP53, SF3B1, BIRC3, NOTCH1, XPO1, EGR2, POT1, NFKBIE, RPS15, FBXW7) associated with inferior prognosis in CLL. In patients with unmutated IGHV (u-IGHV), three cases had ATM VUS (N796H, P1376S, D2870N), including one with concurrent del(11q), 7 had only del(11q), and 20 were without any somatic and/or germline disruption in DDR and CLL-associated genes. Transcriptome analysis of m-IGHV patients with germline missense ATM predicted pathogenic VUS revealed that the highest enrichment scores belonged to mitotic spindle signaling, reactive oxygen species pathway, cholesterol homeostasis, peroxisome, fatty acid metabolism, Wnt/β-catenin signaling, response to UV radiation and oxidative phosphorylation, and genes enriched in targets of MYC, E2F, RUNX1 and CREB1 transcription factors. Analysis of m-IGHV patients with del(11q) revealed similar gene enrichment in mitotic spindle signaling, hypoxia and in the targets of MYC, RUNX1 and CREB1 as in the m-IGHV cohort with ATM VUS, but additionally, genes enriched in TNF-α signaling via NF-κB, apoptosis, and IFN-γ response were detected. Furthermore, two m-IGHV cases had biallelic disruption of TP53; analysis of these patients revealed differentially expressed genes involved in apoptosis, cholesterol homeostasis, oxidative phosphorylation, and targets of MYC and E2F. Analysis of u-IGHV patients with germline missense ATM VUS showed no significant results compared to cases without DDR/CLL gene aberrations, probably due to the low number of cases. Enrichment of genes involved in the p53 pathway, cholesterol homeostasis and MYC targets was observed in u-IGHV patients with only del(11q). Evaluation of another independent CLL cohort is currently underway. Conclusions This preliminary analysis showed a similar impact of rare germline ATM VUS on the CLL transcriptome as del(11q), at least in m-IGHV CLL, which was characterized by differentially expressed genes involved in mitotic spindle and oxidative stress signaling and targets of MYC, RUNX1 and CREB1 transcription factors. These data highlight the need for further investigation and validation of detected transcriptomic changes in a larger cohort, which is ongoing. Grant support: MH CZ - DRO (FNOl, 00098892), IGA LF UP_2022_011

NLRP6 in the Bone Marrow Microenvironment Regulates HSPC Quiescence Via MGP/RUNX2

Inflammatory response in the bone marrow microenvironment affects hematopoietic stem cell (HSC) engraftment and hematopoietic reconstruction. NLR Family Pyrin Domain Containing 6 (NLRP6) involves in multiple signaling pathways to regulate inflammatory response and maintain cell homeostasis. However, the mechanism of NLRP6 in bone marrow microenvironment regulating hematopoiesis remains unclear. To analysis of the effect of NLRP6 deficiency on bone marrow microenvironment and HSC activity, bone marrow chimeras with NLRP6 deficiency in the microenvironment was performed to analyze HSPC quiescence and differentiation in the microenvironment with Nlrp6 deficiency. Single-cell sequencing technology was utilized to analyze the cellular and molecular abnormalities of NLRP6-deficient stromal cells in the bone marrow and to furtherly explore the regulatory mechanism of the NLRP6-deficient microenvironment to resident HSPCs. The results showed that NLRP6 deficiency in bone marrow microenvironment increased proportion of LSK-HSPCs in G0 phase but not effect on the homing of HSPCs. LSK-HSPCs sorted from NLRP6-/- recipients at week 8 after 1st transplantation for 2nd transplantation had higher frequency of CD45.1+ cells in the PBMCs or in total myeloid cells compared with control. NLRP6 deficiency in bone marrow microenvironment reduced CMP and GMP proportion. CMPs sorted from NLRP6-/- recipients at week 8 after 1st transplantation had lower regeneration potential through 2nd transplantation and cell proliferation assay in vitro. Transcriptome analysis of LSK-HSPC, CMP, GMP and MEP from two groups at 8 weeks after transplantation showed that NLRP6 deficiency in the microenvironment modulated cell cycle of HSPCs and is associated with NOD-like receptor signaling pathway. Single-cell sequencing of bone marrow CD45-Ter119- cells showed that NLRP6 deficiency increased CAR cells of bone marrow stroma. The molecule expression of CAR cells (including Cxcl12 and Kitl) contributing to hematopoiesis was upregulated which was associated with MAPK and NOD-like receptor signaling pathway. NLRP6 deletion in the microenvironment upregulated MGP expression of CAR cell which were furtherly supported by western blot and immunofluorescence staining. Recombinant mouse MGP protein (rmMGP) decreased Colony-Forming Units (CFUs) of WT LSK-HSPCs. Single-cell sequencing of LSK-HSPCs at week 8 after 1st transplantation showed that HSCs differentiated into Mpps which separately kept dormant or entered cell cycling. NLRP6 deficiency in the microenvironment markedly increased the quiescent cellular proportion of LSK-HSPCs compared with control, especially Flt3+ CD48- MPPs which were dormant MPPs differentiated early from HSCs. LSK-HSPCs resident in the microenvironment with NLRP6 deletion kept lower transcription of genes about cell cycling and NOD like receptor signaling pathway. Transcription factor Runx2 and its target inhibitors of cell cycle were upregulated and RUNX2 phosphorylation increased by western blot compared with control. In a word, this study suggests that NLRP6 in the bone marrow microenvironment promotes HSPC quiescence induced by RUNX2 through upregulating MGP from CAR cells.

Pathogenic RUNX1 C-Terminal Truncation Mutations Contribute to Differentiation Block and Result in Unique Changes in Transcription and Chromatin Organization

The transcription factor RUNX1 is one of the most frequently mutated genes in hematological disorders, including familial platelet disorder (FPD), predisposition to myeloid dysplastic syndromes (MDS) and acute myeloid leukemia (AML). In addition to a large number of mutations in the N-terminal DNA binding domain (RHD) of RUNX1, 27% of all hematopoietic mutations lie within the C-terminal region beyond the RHD. Previous studies have mainly focused on loss of function RHD mutations while C-terminal mutations are not well characterized and lack "hotspot" mutations. The majority of mutations in the C-terminal region are frameshift and nonsense mutations rather than missense. Some of these mutations are thought to be subject to degradation by nonsense-mediated decay (NMD), emulating loss of RUNX1. Here we demonstrate that RUNX1 C-terminal truncation mutations represent an alternative means of RUNX1 pathogenesis beyond loss of RUNX1 and RHD mutations. To examine the prevalence of RUNX1 C-terminal mutations we first analyzed publicly available data (COSMIC - Sanger Institute) and identified 247 RUNX1 nonsense/frameshift mutations C-terminal of the RHD in patients with blood disorders. Importantly, 76% of these mutations are predicted to escape Nonsense Mediated Decay (NMD) and produce protein products. To study this class of RUNX1 mutation, we introduced the pathogenic RUNX1R320* mutation into the endogenous RUNX1 locus of the human blood cell line K562 using CRISPR-Cas9. We experimentally validated that the RUNX1R320* transcript is not subject to NMD and is expressed at significantly higher levels than the wild-type at both the RNA and protein level. The resulting truncated protein retains the DNA binding RHD but lacks C-terminal domains for binding to other cofactors. To identify biological changes caused by the RUNX1R320* mutation we examined effects on proliferation, differentiation, and DNA damage. Although no significant change in proliferation was observed, megakaryocytic differentiation was impaired. After TPA treatment RUNX1R320* cells failed to fully differentiate and retained high levels of the erythroid marker CD235a. Furthermore, RUNX1R320* showed sensitivity to DNA damage after treatment with etoposide while cell death via apoptosis was not significantly affected. Together these data suggest that C-terminal truncation RUNX1 contributes to differentiation block and an increase in DNA damage sensitivity while evading apoptotic cell signaling pathways. As RUNX1 functions as a master transcriptional regulator of hematopoiesis, we examined how RUNX1 truncations alter transcription and if the effects emulate RUNX1 loss of function mutations by performing RNA-seq with parental, RUNX1 knockdown, and RUNX1R320* knock-in cells. We first identified 2880 significantly differentially expressed genes (DEGs) between wild-type and RUNX1R320*. When compared to RUNX1 knockdown data, we found that the majority of RUNX1R320* DEGs (2052) were unique to RUNX1R320*. This demonstrates that RUNX1R320* results in different transcriptional perturbations than the loss of RUNX1. We next asked whether changes in chromatin confirmation attributed to the observed transcriptional alterations. To study this the newly developed technology Global RNA Interactions with DNA by deep sequencing (GRID-seq) was performed on wild-type and RUNX1R320* cells. Combining these data with H3K27ac and H3K4me3 ChIP-seq data to identify active enhancers and promoters, respectively, we detected 2,891 significantly altered enhancer-promoter (E-P) pairs. Focusing on DEGs with GRID-seq detected enhancer RNA (eRNA), we conducted Gene Ontology (GO) analysis and found enrichment in pathways of chronic myeloid leukemia, regulation of hematopoietic stem cell differentiation, and cell division, revealing the effects of RUNX1R320* on key hematopoietic and myeloid pathways. In summary, we show that RUNX1 C-terminal truncation mutants can escape NMD, contribute to differentiation block, and increase sensitivity to DNA damage. Furthermore, RUNX1R320* affects transcription in a manner distinct from RUNX1 loss. In our GRID-seq analysis, we identify changes in enhancer-promoter interactions that affect key hematological pathways. Our findings demonstrate that pathogenic RUNX1 C-terminal mutations disrupt hematopoietic cell function, transcription, and chromatin in a distinctive fashion.

CD82 Is Sufficient to Uniquely Identify Pluripotent Stem Cell-Derived Hemogenic Endothelium with the Hematopoietic Lineage Potency to Give Rise to Bona Fide Lymphocytes

Chimeric antigen receptor (CAR)-T and natural killer (NK) cells represent compelling opportunities for the treatment of hematological malignancies and solid tumors. However, autologous and donor-derived allogeneic CAR-T and NK cell therapies are limited based on the quantity and quality of the starting engineered cells. We previously described our off-the-shelf induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) and CAR-NK (CAR-iNK) cell platforms that eliminate some of the hurdles associated with patient-derived cells by creating a homogenous cell product that is derived from a renewable bank of iPSC master cell lines. A major challenge in the manufacturing of iPSC derived immune cells is the derivation of hemogenic endothelium (HE), which is the rare and transient direct precursor of hematopoietic cells, making its in vitro specification from iPSCs inefficient and challenging. Furthermore, there is not a reliable marker or set of markers for the identification of HE in culture. HE identification has traditionally relied on detection of the transcription factor RUNX1 and the establishment of reporter cell lines, neither of which are suitable for the manufacture of HE cells destined to become therapeutic immune cells. We have established a cytokine driven protocol for the differentiation of iPSCs that improves the efficiency of the derivation of RUNX1+ HE cells. To identify candidate HE surface markers, we compared iPSC-derived cells containing a high percentage of HE, based on RUNX1 expression, to control differentiated cells containing a low percentage of HE, via a cell surface antibody array. Through the array we identified several surface markers that were expressed by a higher percentage of CD34+ endothelial cells compared to control CD34+ cells. Several of the candidates identified, including CD44, CD61, CD226 and CD143 have been previously identified by other groups as candidate HE markers, thereby validating our screening method. We identified CD82 as a novel candidate HE marker, with expression uniquely restricted to the CD34+ endothelial population and positively correlating with RUNX1 expression (Fig. 1A). Furthermore, we demonstrated via limiting dilution assays that HE is enriched in the CD34+CD43-CD82+ population (HE frequency, 1/38) compared to the CD34+CD43-CD82- population(HE frequency, 1/736) (Fig. 1B). Even more strikingly, isolation of cells based on CD82 alone was sufficient to enrich for HE cells (HE frequency, 1/44) and outcompeted CD34 enrichment alone, suggestive of its unique ability to identify the HE population (Fig. 1B). To determine if CD82+ HE cells could give rise to lymphocytes we carried them forward into T cell differentiation and showed thatthey efficiently gave rise to CAR-iT cells (i.e. >95% CD7+ T lymphocytes). The identification of CD82 for the enrichment of iPSC derived HE is a critical step to the development of definitive hematopoiesis with ability to give rise to all lineages of the hematopoietic compartment and facilitates the scaled and consistent manufacture of high-quality T and NK cells for off-the-shelf therapeutic applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 13063: Clonal Hematopoiesis Associated With TET2 and DNMT3A Gene Driver Mutations Accelerates Aortic Valve Calcification in vitro and in vivo

Calcific aortic valve disease (CAVD) is the most common age-related heart valve disease, with no medical therapy to halt progression. Age is associated with the enrichment of somatic mutations in hematopoietic stem cells leading to clonal hematopoiesis (CH). CH mutations promote inflammation, occur in more than 30% of patients with severe CAVD and CAVD patients harboring CH mutations have a worse prognosis. Single-cell RNA-sequencing of immune cells (n=127120 cells) of CAVD patients with CH-mutations showed strong proinflammatory and M1-like macrophage gene signatures (CD38, CXCL10) along with genes associated with calcification (S100A9, RUNX2, Oncostatin M=OSM) suggesting that CH might be causally involved in CAVD. Indeed, silencing of TET2 or DNMT3A in macrophages in vitro induce the prototypic osteoblastic transcription factor RUNX2 and paracrine acting genes such as OSM and S100A9, which can promote vascular calcification. Secreted factors from TET2 or DNMT3A-silenced macrophages induced osteoblastic differentiation as demonstrated by elevated calcium deposition and genes involved in the RUNX2 signaling pathway (COL1A2, ALP), which could be ablated by silencing of OSM in CH-macrophages. Atheroprone Ldlr -/- mice receiving TET2 -/- bone marrow transplants mice showed increased total calcified area along (1.49-fold) with increased numbers of calcification deposits (2.33-fold) as evidenced by von Kossa staining. Increased myeloid derived OSM and S100A9 was found in the valves of TET2 -/- BMT mice with S100A9 strongly co-occurring with OSM. This study provides insights into the development of incident CAVD for patients with CH mutations and therefore yields plausible interventions for nearly one third of CAVD cases to slow or stop disease progression.

Upregulated Expression of Runt-related Transcription Factor 3 and Interferon Regulatory Factor 4 Genes in Patients With Ankylosing Spondylitis

Background: Ankylosing spondylitis (AS) is a debilitating autoimmune disease presented by chronic inflammation of large joints and central skeleton. The role of various immune cells, including T cell subsets, has been studied in the pathogenesis of AS. Two critical transcription factors, runtrelated transcription factor 3 (RUNX3) and interferon regulatory factor 4 (IRF4), are involved in the differentiation and function of T lymphocytes. This study compared the gene expression level ofRUNX3 and IRF4 between patients with AS and healthy subjects to understand the impact of these factors in the immunopathogenesis of the disease. Methods: Thirty patients with AS and 30 age- and gender-matched healthy individuals were recruited to the study, and expression of RUNX3 and IRF4 genes was evaluated using the reverse transcription polymerase chain reaction (RT-PCR) technique in their peripheral blood. Results: The expression of RUNX3 and IRF4 genes in AS patients was significantly upregulated compared to the healthy controls (P=0.03 and 0.025, respectively). In addition, there was a direct correlation between IRF4 gene expression and bath ankylosing spondylitis global score (BAS-G) (correlation coefficient=0.38, P=0.04) Conclusion: Gene expression of RUNX3 and IRF4 transcription factors involved in T cells’ differentiation and function was increased in AS. These findings might have prognostic and therapeutic value.

Metformin suppresses Oxidative Stress induced by High Glucose via Activation of the Nrf2/HO-1 Signaling Pathway in Type 2 Diabetic Osteoporosis

BackgroundMetformin (MET) is widely used as a first-line hypoglycemic agent for the treatment of type 2 diabetes mellitus (T2DM) and was also confirmed to have a therapeutic effect on type 2 diabetic osteoporosis (T2DOP). However, the potential mechanisms of MET in the treatment of T2DOP are unclear. ObjectiveTo clarify the effect of MET in T2DOP and to explore the potential mechanism of MET in the treatment of T2DOP. MethodsIn vitro, we used MC3T3-E1 cells to study the effects of MET on osteogenic differentiation and anti-oxidative stress injury in a high glucose (Glucose 25 mM) environment. In vivo, we directly used db/db mice as a T2DOP model and assessed the osteoprotective effects of MET by Micro CT and histological analysis. ResultsIn vitro, we found that MET increased ALP activity in MC3T3-E1 cells in a high-glucose environment, promoted the formation of bone mineralized nodules, and upregulated the expression of the osteogenesis-related transcription factors RUNX2, Osterix, and COL1A1-related genes. In addition, MET was able to reduce high glucose-induced reactive oxygen species (ROS) production. In studies on the underlying mechanisms, we found that MET activated the Nrf2/HO-1 signaling pathway and alleviated high-glucose-induced oxidative stress injury. In vivo results showed that MET reduced bone loss and bone microarchitecture destruction in db/db mice. ConclusionOur results suggest that MET can activate the Nrf2/HO-1 signaling pathway to regulate the inhibition of osteogenic differentiation induced by high glucose thereby protecting T2DOP.

Central gene transcriptional regulatory networks shaping monocyte development in bone marrow.

The development of monocytes in bone marrow is a complex process with multiple steps. We used RNA-seq data to analyze the transcriptome profiles in developing stages of monocytes, including hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte–monocyte progenitors (GMPs), and monocytes. We found that genes related to potassium and other cation transmembrane activities and ion binding were upregulated during the differentiation of HSCs into CMPs. Protein transport and membrane surface functional molecules were significantly upregulated in the GMP stage. The CD42RAC and proteasome pathways are significantly upregulated during the development of HSCs into monocytes. Transcription factors Ank1, Runx2, Hmga2, Klf1, Nfia, and Bmyc were upregulated during the differentiation of HSCs into CMPs; Gfi1 and Hmgn2 were highly expressed during the differentiation of CMPs into GMPs; Seventeen transcription factors including Foxo1, Cdkn2d, Foxo3, Ep300, Pias1, Nfkb1, Creb1, Bcl6, Ppp3cb, Stat5b, Nfatc4, Mef2a, Stat6, Ifnar2, Irf7, Irf5, and Cebpb were identified as potentially involved in the development of GMPs into monocytes in mice and humans. In metabolism pathway regulation, HSCs have high glucose, lipid, and nucleic acid metabolism activities; CMPs mainly up regulate the TCA cycle related genes; and GMPs have extremely active metabolisms, with significantly elevated pentose phosphate pathway, TCA cycle, histidine metabolism, and purine metabolism. In the monocyte phase, the tricarboxylic acid (TCA) cycle is reduced, and the anaerobic glycolysis process becomes dominated. Overall, our studies offer the kinetics and maps of gene transcriptional expressions and cell metabolisms during monocyte development in bone marrow.

RUNX1 deficiency cooperates with SRSF2 mutation to induce multilineage hematopoietic defects characteristic of MDS

AbstractMyelodysplastic syndromes (MDSs) are a heterogeneous group of hematologic malignancies with a propensity to progress to acute myeloid leukemia. Causal mutations in multiple classes of genes have been identified in patients with MDS with some patients harboring more than 1 mutation. Interestingly, double mutations tend to occur in different classes rather than the same class of genes, as exemplified by frequent cooccurring mutations in the transcription factor RUNX1 and the splicing factor SRSF2. This prototypic double mutant provides an opportunity to understand how their divergent functions in transcription and posttranscriptional regulation may be altered to jointly promote MDS. Here, we report a mouse model in which Runx1 knockout was combined with the Srsf2 P95H mutation to cause multilineage hematopoietic defects. Besides their additive and synergistic effects, we also unexpectedly noted a degree of antagonizing activity of single mutations in specific hematopoietic progenitors. To uncover the mechanism, we further developed a cellular model using human K562 cells and performed parallel gene expression and splicing analyses in both human and murine contexts. Strikingly, although RUNX1 deficiency was responsible for altered transcription in both single and double mutants, it also induced dramatic changes in global splicing, as seen with mutant SRSF2, and only their combination induced missplicing of genes selectively enriched in the DNA damage response and cell cycle checkpoint pathways. Collectively, these data reveal the convergent impact of a prototypic MDS-associated double mutant on RNA processing and suggest that aberrant DNA damage repair and cell cycle regulation critically contribute to MDS development.

Inducible general knockout of Runx3 profoundly reduces pulmonary cytotoxic CD8+ T cells with minimal effect on outcomes in mice following influenza infection.

Respiratory viruses pose a continuing and substantive threat to human health globally. Host innate and adaptive immune responses are the critical antiviral defense mechanisms to control viral replication and spread. The present study is designed to determine the role of transcription factor Runx3 in the host immune response to influenza A virus (IAV) infection. As Runx3 is required for embryonic development, we generated an inducible Runx3 global knockout (KO) mouse model and found that Runx3 KO in adult C57BL/6 mice minimally affected thymic function under normal conditions and survival was at least 250 days post Runx3 deletion. We applied the mouse model to IAV infection and found that Runx3 KO resulted in a huge reduction (>85%) in numbers of total and antigen-specific pulmonary CD8+ cytotoxic T cells during IAV infection, while it had a minor effect on pulmonary generation of CD4+ T cells. To our surprise, this general KO of Runx3 did not significantly alter viral clearance and animal survival following IAV infection. Interestingly, we found that Runx3 KO significantly increased the numbers of pulmonary innate immune cells such as macrophages and neutrophils and the production of pro-inflammatory cytokines during IAV infection. We further found that Runx3 was strongly detected in CCR2+ immune cells in IAV-infected mouse lungs and was induced in activated macrophages and dendritic cells (DCs). As pulmonary CD8+ cytotoxic T cells play a central role in the clearance of IAV, our findings suggest that Runx3 KO may enhance host innate immunity to compensate for the loss of pulmonary CD8+ cytotoxic T cells during IAV infection.

Emerging Therapeutic Targets for Heart Failure.

Heart failure is a global epidemic that affects at least 26 million individuals globally and is becoming more prevalent. Despite advances in treatment strategies, survival and symptom management in individuals with heart failure remain exceptionally low. This review discusses emerging targets for the treatment of heart failure. Recently, a number of targets are being investigated as prospective treatment possibilities for heart failure. These include targets like Runx1 transcription factor (RUNX1), milk fact globule-EFG factor 8 (MFGE8) protein and enzymes such as neuraminidase 1 (NEU1), G protein-coupled receptor kinase 5 (GRK5), G protein-coupled oestrogen receptor 1 (GPER1), urotensin-II receptor (UTR), cluster of differentiation 47 (CD47) and relaxin receptor 1 (RXFP1). On a worldwide level, heart failure is a developing epidemic with substantial morbidity and death. The number of individuals diagnosed with chronic heart failure is rising, and it is anticipated to surge by 46% by 2030. Appropriate heart failure treatment can have the greatest influence on prolonging patients' lives in the coming year. Targets discussed in this review may provide new therapeutic approaches for the treatment of heart failure.

Sodium butyrate ameliorates thiram-induced tibial dyschondroplasia and gut microbial dysbiosis in broiler chickens

Thiram is a dithiocarbamate pesticide widely used in agriculture as a fungicide for storing grains to prevent fungal diseases. However, its residues have threatened the safety of human beings and the stability of the ecosystem by causing different disease conditions, e.g., tibial dyschondroplasia (TD), which results in a substantial economic loss for the poultry industry. So, the research on TD has a great concern for the industry and the overall GDP of a country. In current study, we investigated whether different concentrations (300, 500, and 700 mg/kg) of sodium butyrate alleviated TD induced under acute thiram exposure by regulating osteogenic gene expression, promoting chondrocyte differentiation, and altering the gut microbial community. According to the findings, sodium butyrate restored clinical symptoms in broilers, improved growth performance, bone density, angiogenesis, and chondrocyte morphology and arrangement. It could activate the signal transduction of the Wnt/β-catenin pathway, regulate the expression of GSK-3β and β-catenin, and further promote the production of osteogenic transcription factors Runx2 and OPN for restoration of lameness. In addition, the 16S rRNA sequencing revealed a significantly different community composition among the groups. The TD group increased the abundance of the harmful bacteria Proteobacteria, Subdoligranulum, and Erysipelatoclostridium. The sodium butyrate enriched many beneficial bacteria, such as Bacteroidetes, Verrucomicrobia, Faecalibacterium, Barnesiella, Rikenella, and Butyricicoccus, etc., especially at the concentration of 500 mg/kg. The mentioned concentration significantly limited the intestinal disorders under thiram exposure, and restored bone metabolism.

Enamel biomineralization under the effects of indomethacin and celecoxib non-steroidal anti-inflammatory drugs

The aim of this study was to explore the effects of nonsteroidal anti-inflammatory drugs on biomineralization of enamel. Sixty C57Bl6 male mice were used, which were assigned into three groups: celecoxib (n = 20) or indomethacin (n = 20) treatment for a period of 28 days or received no medication (control group, n = 20). Visual inspection and microcomputed tomography were used to analyze enamel morphology. Scanning electron microscopy–Energy dispersive X-ray and Knoop microhardness test were used to quantify chemical element content (Ca, P, C, O) and enamel microhardness, respectively. Tissues were collected to investigate the synthesis, activity or nuclear translocation of metalloproteinase-20, transcription factor Runx2, dentin sialoprotein and cyclooxygenase-2 enzyme by means of immunohistochemistry, in situ zymography and indirect immunofluorescence. Treatment with indomethacin and celecoxib reduced the Ca and P content, microhardness and mineral density in enamel. Treatment with nonsteroidal anti-inflammatory drugs caused an accumulation of metalloproteinase-20 and overall increased enzymatic activity in enamel matrix, while the synthesis of the transcription factor Runx2 was inhibited by these drugs. Interestingly, indomethacin inhibited Runx2 translocation to the nucleus whereas celecoxib did not. Those findings show that non-steroidal anti-inflammatory drugs impact the enamel biomineralization and could be involved in the etiology tooth enamel defects if used during the period of tooth formation and mineralization.